Epidermal Growth Factor Receptor (EGFR) is a cell surface protein that binds to its ligand, epidermal growth factor (EGF) and related ligands, and controls cell growth and differentiation as a result of this receptor-ligand interaction. EGFR is a member of the tyrosine kinase superfamily of signaling molecules that participate in ligand-mediated, cell proliferation mechanisms. Like other members of the receptor tyrosine kinase family, EGFR is an oncogene that has an important role in tumorigenesis. Mutations in EGFR are associated with a variety of cancers, including, for example, adenocarcinoma, adrenocortical cancer, biliary cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gall bladder cancer, gastric cancer, glioma, glioblastoma, and glioblastoma multiforme, head and neck cancer, lung cancer, pancreatic cancer, and salivary cancer.
Because of its importance in cell proliferation and its involvement as an oncogenic agent in cancers, considerable research interest has focused on understanding the mechanism of EGFR activation and signaling. The protein encoded by the EGFR gene is a transmembrane glycoprotein that possesses an extracellular ligand binding domain, a transmembrane domain, and an intracellular kinase domain. Upon binding to EGF or structurally related EGF-like molecules, EGFR undergoes protein dimerization and tyrosine autophosphorylation by the intracellular kinase domain that leads to receptor activation. Activated EGFR phosphorylates other cytoplasmic substrates as part of the signaling cascade, resulting in numerous changes in gene expression and cellular physiology, including anti-apoptosis and increased cell proliferation.
Mutations in the EGFR gene can lead to overexpression and/or inappropriate activation of the EGFR protein and potentially unchecked cell proliferation. A significant percentage of epithelial cancers are associated with mutation, rearrangement, and/or ectopic regulation of the EGFR gene. In addition, amplification of the EGFR gene occurs in many cancers. For example, EGFR gene amplification arises in at least 40% of malignant gliomas.
Although a number of oncogenic point mutations occur in the EGFR gene, the constitutively active deletion mutation known as EGFR variant III (EGFR(v3)) appears to be the most common activating mutation found to date. For example, EGFR(v3) is the most common deletion mutant expressed in malignant gliomas and has been reported to occur in almost half of such tumors. EGFR(v3) contains an in-frame deletion of exons 2 through 7 corresponding to amino acids 6 through 273. This gene produces a 140- to 145-kDa receptor with unique epitopes. Deletion of this coding information maps to the extracellular ligand-binding domain of EGFR(v3) and impacts the binding affinity of EGFR(v3) for EGF and related ligands. Although EGFR(v3) cannot bind ligand with high affinity, it is constitutively autophosphorylated. Consequently, EGFR(v3) is of substantial interest due to its effects on signal transduction and as a potential tumor-specific target.
EGFR is an attractive target for anticancer therapeutic agent development. EGFR gene amplification status in a given tumor may indicate whether cancer cells will respond to therapeutics directed to EGFR. And many of the tumors harboring an amplified EGFR gene produce a mutant EGFR protein. Thus, prognosis and therapeutic response may also depend upon whether the expressed EGFR gene is mutant or wild type.
In situ methods provide a powerful and sensitive means for detecting the presence of tumor-specific antigens in biological specimens. Several challenges remain for detecting in situ the presence of the EGFR(v3) gene products in tumors. The EGFR(v3) mRNA is difficult to detect in situ with reverse transcriptase-based PCR techniques, owing to the highly degraded state of RNA in formalin-fixed paraffin-embedded specimens. Antibodies directed against the EGFR(v3) protein are also poor for use in immunohistochemistry methodologies. Thus, the existing in situ methods are not amenable for efficiently detecting EGFR(v3) status in tumors.